Clinica Chimica Acta 446 (2015) 73–75
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Case report
Preliminary evaluation of an improved enzymatic assay method for measuring potassium concentrations in serum Eri Ota a, Shin-ichi Sakasegawa b,⁎, Shigeru Ueda b, Kenji Konishi b, Masaru Akimoto a, Takiko Tateishi a, Miki Kawano a, Eisaku Hokazono a, Yuzo Kayamori a a b
Division of Biological Science and Technology, Department of Health Science, Faculty of Medical Sciences, Kyushu University, Japan Asahi Kasei Pharma Corporation, Japan
a r t i c l e
i n f o
Article history: Received 6 December 2014 Received in revised form 25 February 2015 Accepted 2 March 2015 Available online 16 April 2015 Keywords: Potassium Inosine 5′-monophosphate dehydrogenase Enzymatic assay method
a b s t r a c t Background: K+ has important physiological functions. K+ concentrations in serum are generally determined using ion-selective electrodes (ISEs), though measurement using reagents in aqueous medium is also useful. Methods: K+ concentrations were measured using recombinant inosine 5′-monophosphate dehydrogenase + + (IMPDH), which was activated only by K+ and NH+ 4 . Exogenous NH4 and endogenous NH4 were eliminated using glutamine synthase. Results: Regression analysis of the enzymatic assay (y) vs. the ISE method (x) gave the following relation: y = 1.03x + 0.09 (n = 54, Sy,x = 0.06 mmol/l). The linear range was up to 12 mmol/l when 1 U/ml IMPDH was used. Conclusion: Advantages of the proposed assay method are: (i) the measured range is wider than that of existing enzymatic methods; (ii) the conditions for K+ determination can be maintained constant, regardless of the amount of NH+ 4 in the analyte and reagents; and (iii) the elimination system is simpler because the recombinant IMPDH is stimulated by only K+ and NH+ 4 and is unaffected by biological materials. © 2015 Published by Elsevier B.V.
1. Introduction K+ has important physiological functions, some in common with other electrolytes. Serum K+ concentrations are generally determined using ion-selective electrodes (ISEs). However, there is a desire for reagents with which to measure K+ concentrations in aqueous medium, because ISE is generally an optional equipment and requires a lot of maintenance. To date, three enzymatic methods for measuring K+ in aqueous medium have been developed [1–3]. We report here a novel improved method that uses recombinant inosine 5′-monophosphate dehydrogenase (IMPDH, EC 1. 1. 1. 205) from Bacillus subtilis. Wild+ + type IMPDH from B. subtilis is stimulated by Tl+, K+, NH+ 4 , Rb , Cs , Na+ or Li+ [4], but we produced a recombinant form activated only by K+ or NH+ 4 [5].
using an electrode attached to a Hitachi biochemical analyzer. Recombinant IMPDH was previously developed in our laboratory [5]. Glutamine synthetase (GS, EC 6.3.1.2) was obtained from Asahi Kasei Pharma. All other chemicals were purchased from Sigma Chemical Co, Oriental Yeast or Wako Pure Chemicals. The enzymatic assay consists of two independent reactions run (Fig. 1) using two separate reaction mixtures A and B. In reaction #1, NH+ 4 in the sample and reagents was ligated to Lglutamate to form L-glutamine using GS in the presence of ATP and Mg2+. Moreover, reaction #1 was also ongoing in reaction mixtureB, so that the NH+ 4 elimination proceeded continuously at all times.
Reaction #1 L-glutamate + NH4+
2. Materials and methods All procedures were approved by the Ethics Committee in Kyushu University, and all sera were collected from patients at Kyushu University Hospital after receiving informed consent. Multiple batches of reagent and multiple recalibrations were performed during the study. Enzymatic assays were performed using a Hitachi 7170s automated analyzer (Hitachi). For comparison, the conventional ISE method was performed ⁎ Corresponding author at: Asahi Kasei Pharma Corporation, Shizuoka 410-2321, Japan. E-mail address: [email protected] (S. Sakasegawa).
http://dx.doi.org/10.1016/j.cca.2015.03.042 0009-8981/© 2015 Published by Elsevier B.V.
GS Mg 2+ ATP ADP
L-glutamine + phosphate
Reaction #2 Inosine 5’monophosphate
IMPDH K+ NAD
Xanthosine 5’monophosphate
NADH
Fig. 1. Reaction scheme for measuring K+ concentrations. Reaction #1 is for elimination of + NH+ 4 . Reaction #2 is for measuring K .
E. Ota et al. / Clinica Chimica Acta 446 (2015) 73–75
IMPDH method (mmol/L)
74
8 6 4 2 0
(B) ΔAbs/min
Abs at 340 nm
10 mmol/l KCl (A)
Time (min)
KCl (mmol/l)
(C)
ISE (mmol/l)
Fig. 2. (A) Time courses of the K+ (0 to 10 mmol/l in samples) assays. Shown are time-dependent changes in the absorbance at 340 nm during the reactions. The IMPDH concentration is 2 U/ml. (B) Calibration curves for the K+ assays. Shown are the relationships between the absorbance increases and IMPDH concentrations in reaction mixture B: open circles, 1.0 U/ml; filled circles, 1.5 U/ml; open triangles, 2.0 U/ml; filled triangles, 2.5 U/ml. Equations for the linear-regression lines are y = 0.0050x + 0.0256 (r = 0.9997), y = 0.0072x + 0.0386 (r = 0.9996), y = 0.0096x + 0.0499 (r = 0.9994), and y = 0.0115x + 0.0649 (r = 0.9995), respectively. (C) Regression analysis of the proposed enzymatic assay (y) vs. the ion-selective electrode (ISE) method (x). The Deming regression equation was y = 1.03 (95% CI, 0.99 to 1.11) x + 0.09 (−0.23 to 0.29) (r = 0.983, Sy,x = 0.06 mmol/l).
Reaction #2 was the IMPDH reaction, which was activated by K+. Reaction mixture A contained 50 mmol/l TrisHCl (pH 8.5), 5 mmol/l inosine 5′-monophosphate (IMP), 5 mmol/l NAD, 1 mmol/l MgCl2, 1 mmol/l ATP, 20 mmol/l L-glutamate Na and 2 U/ml GS. Reaction mixture B contained 50 mmol/l Tris–HCl (pH 8.5), 1 U/ml IMPDH, 4 mmol/l thioglycerol, 1 mmol/l MgCl2, 1 mmol/l ATP, 20 mmol/l L-glutamate Na and 2 U/ml GS. In each automated assay, 150 μl of mixture A was incubated with 5 μl of sample for 5 min at 37 °C, after which 50 μl of mixture B was added. About 3 min after addition of mixture B, NADH formation was measured for approximately 1 min based on the increasing absorbance at 340 nm. The assay mode was Rate-A, and a 2-point calibration was used. Unless otherwise noted, calibrators contained 0 or 9.0 mmol/l K+ (achieved by adding KCl to distilled water). Human serum samples were frozen at − 20 °C until just before the assays. The limit of blank (LoB) and limit of detection (LoD) were calculated from 20 measurements made over 4 days, and the type I error was set to 5% [6]. A lower limit of quantification (LoQ) was obtained by plotting the CV (determined from 20 measurements made over 4 days at 10 concentrations around the LoQ) against the mean concentration and then estimating the concentration at which the CV did not exceed 5%. Pooled serum samples spiked with bilirubin F, bilirubin C, hemolytic hemoglobin or chyle (Interference check A+, Sysmex) were assayed for the interference test. 3. Results and discussion IMPDH was activated by K+ in a concentration-dependent fashion (Fig. 2(A)). In addition, the sensitivity and NADH production rates (ΔAbs/min) at different K+ concentrations reflected the IMPDH
concentration (Fig. 2(B)). The calibration curve was linear up to at least 12 mmol/l when 1 U/ml IMPDH was used: y = 0.0050x + 0.0256 (r = 0.9997). However, the linear range could be adjusted by changing the amount of IMPDH in reaction mixture B; e.g., a wider linear range was obtained by reducing amount of IMPDH, though there was a reduction in sensitivity (Fig. 2(B)). NH+ 4 , which also activates IMPDH and could cause a positive error, was eliminated using GS. The amount of GS used depended on the + expected NH + 4 concentration in specimen; e.g., 1.5 mmol/l NH 4 was eliminated by 2 U/ml GS. Although the reference interval of ammonium is 11–32 μmol/l (adults [7]), the higher concentration of GS should be needed for samples from hyperammonemia and/or containing ammonium heparin as an anticoagulant. In Fig. 2(C), K+ concentrations in serum samples from 54 volunteers were measured using the proposed enzymatic method (y) and plotted against values obtained using the ISE method (x). The plot gave a best-fit Deming regression equation of y = 1.03 (95% CI, 0.99 to 1.11) x + 0.09 (−0.23 to 0.29) (r = 0.983, Sy,x = 0.06 mmol/l). The imprecision of ISE was reported to be 1.28 CV% (daily) [8]. Although the cost of enzymatic method is probably higher than that of ISE, the enzymatic method could be used by automated analyzers without ISE. The within-run and between-run reproducibility of the method is summarized in Table 2. The imprecision of the method may be limited compared to ISE, because the between-run imprecision is based on 5 days only. The LoB, LoD and LoQ were 0.04, 0.08 and 0.33 mmol/l, respectively. Recoveries of K+ added to sera were 99.4 ± 0.3, 99.3 ± 0.4, 99.1 ± 0.5 and 94.3 ± 0.6% at 0.5, 1.0, 4.0 and 12 mmol/l K+, respectively (n = 4). No interference resulted from addition of 185 g/l bilirubin F,
Table 1 Comparison of the four enzymatic methods. Enzyme to measure K+ Pyruvate kinase from Bacillus stearothermophilus Tryptophanase from Escherichia coli Activators Inhibitors
2+ K+, Na+, NH+ and Mg2+ 4 , Mn None
Pyridoxal 5-phosphate, K+, and NH+ K+ and NH+ 4 4 Na+ (moderately) Ca2+ (strong)
Substances to enhance selectivityb Cryptand (Na+), Li+ (Na+) Glu DHc (NH+ 4 ) Coupling enzyme to measure K+ Glu DHc Monitored signal Decreasing NADH
Glu DHc (NH+ 4 ) Na+ (Na+) c Glu DH Decreasing NADPH
Linearity Reference
Up to 7.00 mmol/l [2]
a b c d e
1.0 to 8.0 mmol/l [1]
Inosine 5′-monophosphate dehydrogenase. Substances used to eliminate the influence of the ions in parentheses. Glutamate dehydrogenase (EC 1.4.1.4). Glutamine synthetase. When there was 1 U/ml IMPDH in reaction mixture B.
Urea amidolyase
Glu DHc (NH+ 4 ) GEDTA (Ca2+) c Glu DH Decreasing NADPH Up to 8.00 mmol/l [3]
IMPDHa from Bacillus subtilis K+ and NH+ 4 Nucleotides (Ki = 0.15–5.0 mM) GSd (NH+ 4 ) None Increasing NADH Up to 12 mmol/le [5] and this study
E. Ota et al. / Clinica Chimica Acta 446 (2015) 73–75 Table 2 Within-run and between-run reproducibility of the IMPDH method.
Within-run (n = 5)
Between-run (5 consecutive days)
a b
K+, mmol/l
CV, %
3.06a 4.00a 4.95a 12.7b 3.06a 4.00a 4.95a 12.7b
1.18 0.69 0.51 0.97 2.00 1.28 1.58 0.87
Pooled serum samples containing the indicated K+ concentration were used. Pooled sera sample spiked with KCl was used.
75
By contrast, in all the other enzymatic methods, the cofactor NAD(P)H is shared by the K+-measuring and NH+ 4 elimination reactions; i.e., NAD(P)H is consumed by the NH+ 4 elimination reaction, and the decreasing concentration of the remaining NAD(P)H is monitored to calculate K+ concentrations (Table 1). This means that the K+ reaction rate may be affected by the amount of NH+ 4 in samples and reagents. (iii) The elimination system in the proposed method was simple because IMPDH was only stimulated by K+ and NH+ 4 , and was unaffected by other biological materials. By contrast, in all other enzymatic methods, one or more elimination systems besides NH+ 4 are necessary for Ca2+ or Na+ in the analyte. References
202 g/l bilirubin C, 5.00 g/l hemolytic hemoglobin, 2 mmol/l NH4Cl, 3 mmol/l LiCl, 0.05 mmol/l MnCl2, 200 mmol/l NaCl, 10 mmol/l CaCl2, or 0.1 mmol/l ZnSO4, as well as turbidity up to 1560 hormadin turbidity units. The reagents appear to be stable for at least 7 days at 4 to 8 °C and 1 month at −20 °C, as they retained their initial sensitivity under those conditions. We have developed a novel method for using IMPDH to measure K+ in serum based on increases in NADH, which is unlike all other reported enzymatic methods (Table 1) [1–3]. This proposed assay has several advantages over other enzymatic methods. (i) Because increases in NADH are being monitored, the measured range is wider than that of other methods, where the optical limits of the colorimeters reduce the range of NAD(P)H concentrations that can be measured (Tables 1 and 2). (ii) In the proposed method, the reagent conditions for K+ determination could be maintained constant, because the substrate and cofactor for the IMPDH reaction were unaffected by the NH+ 4 elimination reaction.
[1] Berry MN, Mazzachi RD, Pejakovic M, Peake MJ. Enzymatic determination of potassium in serum. Clin Chem 1989;35:817–20. [2] Kimura S, Asari S, Hayashi S, Yamaguchi Y, Fushimi R, Amino N, et al. New enzymatic method with tryptophanase for determining potassium in serum. Clin Chem 1992;38: 44–7. [3] Kimura S, Iyama S, Yamaguchi Y, Hayashi S, Fushimi R, Amino N. New enzymatic assay with urea amidolyase for determining potassium in serum. Ann Clin Biochem 1997;34:384–8. [4] Wu TW, Scrimgeour KG. Properties of inosinic acid dehydrogenase from Bacillus subtilis. II. Kinetic properties. Can J Biochem 1973;51:1391–8. [5] Ota H, Yasuda Y, Sakasegawa S, Imamura S, Tamura T. A novel enzymatic method for measuring mizoribine phosphate levels in serum. J Biosci Bioeng 2008;106:511–4. [6] Tholen DW, Linnet K, Kondratovich M, Armbruster DA, Garrett PE, Jones RL, et al. NCCLS document EP17-A: protocols for determination of limits of detection and limits of quantitation; approved guideline, vol. 24. Pennsylvania: National Committee for Clinical Laboratory Standards; 2004. [7] http://www.acb.org.uk/whatwedo/science/AMALC.aspx. [8] Kinoshita S, Toyofuku M, Iida H, Wakiyama M, Kurihara M, Nakahara M, et al. Standardization of laboratory data and establishment of reference intervals in the Fukuoka Prefecture: a Japanese perspective. Clin Chem Lab Med 2001;39:256–62.
Contents lists available at ScienceDirect
Clinica Chimica Acta journal homepage: www.elsevier.com/locate/clinchim
Case report
Preliminary evaluation of an improved enzymatic assay method for measuring potassium concentrations in serum Eri Ota a, Shin-ichi Sakasegawa b,⁎, Shigeru Ueda b, Kenji Konishi b, Masaru Akimoto a, Takiko Tateishi a, Miki Kawano a, Eisaku Hokazono a, Yuzo Kayamori a a b
Division of Biological Science and Technology, Department of Health Science, Faculty of Medical Sciences, Kyushu University, Japan Asahi Kasei Pharma Corporation, Japan
a r t i c l e
i n f o
Article history: Received 6 December 2014 Received in revised form 25 February 2015 Accepted 2 March 2015 Available online 16 April 2015 Keywords: Potassium Inosine 5′-monophosphate dehydrogenase Enzymatic assay method
a b s t r a c t Background: K+ has important physiological functions. K+ concentrations in serum are generally determined using ion-selective electrodes (ISEs), though measurement using reagents in aqueous medium is also useful. Methods: K+ concentrations were measured using recombinant inosine 5′-monophosphate dehydrogenase + + (IMPDH), which was activated only by K+ and NH+ 4 . Exogenous NH4 and endogenous NH4 were eliminated using glutamine synthase. Results: Regression analysis of the enzymatic assay (y) vs. the ISE method (x) gave the following relation: y = 1.03x + 0.09 (n = 54, Sy,x = 0.06 mmol/l). The linear range was up to 12 mmol/l when 1 U/ml IMPDH was used. Conclusion: Advantages of the proposed assay method are: (i) the measured range is wider than that of existing enzymatic methods; (ii) the conditions for K+ determination can be maintained constant, regardless of the amount of NH+ 4 in the analyte and reagents; and (iii) the elimination system is simpler because the recombinant IMPDH is stimulated by only K+ and NH+ 4 and is unaffected by biological materials. © 2015 Published by Elsevier B.V.
1. Introduction K+ has important physiological functions, some in common with other electrolytes. Serum K+ concentrations are generally determined using ion-selective electrodes (ISEs). However, there is a desire for reagents with which to measure K+ concentrations in aqueous medium, because ISE is generally an optional equipment and requires a lot of maintenance. To date, three enzymatic methods for measuring K+ in aqueous medium have been developed [1–3]. We report here a novel improved method that uses recombinant inosine 5′-monophosphate dehydrogenase (IMPDH, EC 1. 1. 1. 205) from Bacillus subtilis. Wild+ + type IMPDH from B. subtilis is stimulated by Tl+, K+, NH+ 4 , Rb , Cs , Na+ or Li+ [4], but we produced a recombinant form activated only by K+ or NH+ 4 [5].
using an electrode attached to a Hitachi biochemical analyzer. Recombinant IMPDH was previously developed in our laboratory [5]. Glutamine synthetase (GS, EC 6.3.1.2) was obtained from Asahi Kasei Pharma. All other chemicals were purchased from Sigma Chemical Co, Oriental Yeast or Wako Pure Chemicals. The enzymatic assay consists of two independent reactions run (Fig. 1) using two separate reaction mixtures A and B. In reaction #1, NH+ 4 in the sample and reagents was ligated to Lglutamate to form L-glutamine using GS in the presence of ATP and Mg2+. Moreover, reaction #1 was also ongoing in reaction mixtureB, so that the NH+ 4 elimination proceeded continuously at all times.
Reaction #1 L-glutamate + NH4+
2. Materials and methods All procedures were approved by the Ethics Committee in Kyushu University, and all sera were collected from patients at Kyushu University Hospital after receiving informed consent. Multiple batches of reagent and multiple recalibrations were performed during the study. Enzymatic assays were performed using a Hitachi 7170s automated analyzer (Hitachi). For comparison, the conventional ISE method was performed ⁎ Corresponding author at: Asahi Kasei Pharma Corporation, Shizuoka 410-2321, Japan. E-mail address: [email protected] (S. Sakasegawa).
http://dx.doi.org/10.1016/j.cca.2015.03.042 0009-8981/© 2015 Published by Elsevier B.V.
GS Mg 2+ ATP ADP
L-glutamine + phosphate
Reaction #2 Inosine 5’monophosphate
IMPDH K+ NAD
Xanthosine 5’monophosphate
NADH
Fig. 1. Reaction scheme for measuring K+ concentrations. Reaction #1 is for elimination of + NH+ 4 . Reaction #2 is for measuring K .
E. Ota et al. / Clinica Chimica Acta 446 (2015) 73–75
IMPDH method (mmol/L)
74
8 6 4 2 0
(B) ΔAbs/min
Abs at 340 nm
10 mmol/l KCl (A)
Time (min)
KCl (mmol/l)
(C)
ISE (mmol/l)
Fig. 2. (A) Time courses of the K+ (0 to 10 mmol/l in samples) assays. Shown are time-dependent changes in the absorbance at 340 nm during the reactions. The IMPDH concentration is 2 U/ml. (B) Calibration curves for the K+ assays. Shown are the relationships between the absorbance increases and IMPDH concentrations in reaction mixture B: open circles, 1.0 U/ml; filled circles, 1.5 U/ml; open triangles, 2.0 U/ml; filled triangles, 2.5 U/ml. Equations for the linear-regression lines are y = 0.0050x + 0.0256 (r = 0.9997), y = 0.0072x + 0.0386 (r = 0.9996), y = 0.0096x + 0.0499 (r = 0.9994), and y = 0.0115x + 0.0649 (r = 0.9995), respectively. (C) Regression analysis of the proposed enzymatic assay (y) vs. the ion-selective electrode (ISE) method (x). The Deming regression equation was y = 1.03 (95% CI, 0.99 to 1.11) x + 0.09 (−0.23 to 0.29) (r = 0.983, Sy,x = 0.06 mmol/l).
Reaction #2 was the IMPDH reaction, which was activated by K+. Reaction mixture A contained 50 mmol/l TrisHCl (pH 8.5), 5 mmol/l inosine 5′-monophosphate (IMP), 5 mmol/l NAD, 1 mmol/l MgCl2, 1 mmol/l ATP, 20 mmol/l L-glutamate Na and 2 U/ml GS. Reaction mixture B contained 50 mmol/l Tris–HCl (pH 8.5), 1 U/ml IMPDH, 4 mmol/l thioglycerol, 1 mmol/l MgCl2, 1 mmol/l ATP, 20 mmol/l L-glutamate Na and 2 U/ml GS. In each automated assay, 150 μl of mixture A was incubated with 5 μl of sample for 5 min at 37 °C, after which 50 μl of mixture B was added. About 3 min after addition of mixture B, NADH formation was measured for approximately 1 min based on the increasing absorbance at 340 nm. The assay mode was Rate-A, and a 2-point calibration was used. Unless otherwise noted, calibrators contained 0 or 9.0 mmol/l K+ (achieved by adding KCl to distilled water). Human serum samples were frozen at − 20 °C until just before the assays. The limit of blank (LoB) and limit of detection (LoD) were calculated from 20 measurements made over 4 days, and the type I error was set to 5% [6]. A lower limit of quantification (LoQ) was obtained by plotting the CV (determined from 20 measurements made over 4 days at 10 concentrations around the LoQ) against the mean concentration and then estimating the concentration at which the CV did not exceed 5%. Pooled serum samples spiked with bilirubin F, bilirubin C, hemolytic hemoglobin or chyle (Interference check A+, Sysmex) were assayed for the interference test. 3. Results and discussion IMPDH was activated by K+ in a concentration-dependent fashion (Fig. 2(A)). In addition, the sensitivity and NADH production rates (ΔAbs/min) at different K+ concentrations reflected the IMPDH
concentration (Fig. 2(B)). The calibration curve was linear up to at least 12 mmol/l when 1 U/ml IMPDH was used: y = 0.0050x + 0.0256 (r = 0.9997). However, the linear range could be adjusted by changing the amount of IMPDH in reaction mixture B; e.g., a wider linear range was obtained by reducing amount of IMPDH, though there was a reduction in sensitivity (Fig. 2(B)). NH+ 4 , which also activates IMPDH and could cause a positive error, was eliminated using GS. The amount of GS used depended on the + expected NH + 4 concentration in specimen; e.g., 1.5 mmol/l NH 4 was eliminated by 2 U/ml GS. Although the reference interval of ammonium is 11–32 μmol/l (adults [7]), the higher concentration of GS should be needed for samples from hyperammonemia and/or containing ammonium heparin as an anticoagulant. In Fig. 2(C), K+ concentrations in serum samples from 54 volunteers were measured using the proposed enzymatic method (y) and plotted against values obtained using the ISE method (x). The plot gave a best-fit Deming regression equation of y = 1.03 (95% CI, 0.99 to 1.11) x + 0.09 (−0.23 to 0.29) (r = 0.983, Sy,x = 0.06 mmol/l). The imprecision of ISE was reported to be 1.28 CV% (daily) [8]. Although the cost of enzymatic method is probably higher than that of ISE, the enzymatic method could be used by automated analyzers without ISE. The within-run and between-run reproducibility of the method is summarized in Table 2. The imprecision of the method may be limited compared to ISE, because the between-run imprecision is based on 5 days only. The LoB, LoD and LoQ were 0.04, 0.08 and 0.33 mmol/l, respectively. Recoveries of K+ added to sera were 99.4 ± 0.3, 99.3 ± 0.4, 99.1 ± 0.5 and 94.3 ± 0.6% at 0.5, 1.0, 4.0 and 12 mmol/l K+, respectively (n = 4). No interference resulted from addition of 185 g/l bilirubin F,
Table 1 Comparison of the four enzymatic methods. Enzyme to measure K+ Pyruvate kinase from Bacillus stearothermophilus Tryptophanase from Escherichia coli Activators Inhibitors
2+ K+, Na+, NH+ and Mg2+ 4 , Mn None
Pyridoxal 5-phosphate, K+, and NH+ K+ and NH+ 4 4 Na+ (moderately) Ca2+ (strong)
Substances to enhance selectivityb Cryptand (Na+), Li+ (Na+) Glu DHc (NH+ 4 ) Coupling enzyme to measure K+ Glu DHc Monitored signal Decreasing NADH
Glu DHc (NH+ 4 ) Na+ (Na+) c Glu DH Decreasing NADPH
Linearity Reference
Up to 7.00 mmol/l [2]
a b c d e
1.0 to 8.0 mmol/l [1]
Inosine 5′-monophosphate dehydrogenase. Substances used to eliminate the influence of the ions in parentheses. Glutamate dehydrogenase (EC 1.4.1.4). Glutamine synthetase. When there was 1 U/ml IMPDH in reaction mixture B.
Urea amidolyase
Glu DHc (NH+ 4 ) GEDTA (Ca2+) c Glu DH Decreasing NADPH Up to 8.00 mmol/l [3]
IMPDHa from Bacillus subtilis K+ and NH+ 4 Nucleotides (Ki = 0.15–5.0 mM) GSd (NH+ 4 ) None Increasing NADH Up to 12 mmol/le [5] and this study
E. Ota et al. / Clinica Chimica Acta 446 (2015) 73–75 Table 2 Within-run and between-run reproducibility of the IMPDH method.
Within-run (n = 5)
Between-run (5 consecutive days)
a b
K+, mmol/l
CV, %
3.06a 4.00a 4.95a 12.7b 3.06a 4.00a 4.95a 12.7b
1.18 0.69 0.51 0.97 2.00 1.28 1.58 0.87
Pooled serum samples containing the indicated K+ concentration were used. Pooled sera sample spiked with KCl was used.
75
By contrast, in all the other enzymatic methods, the cofactor NAD(P)H is shared by the K+-measuring and NH+ 4 elimination reactions; i.e., NAD(P)H is consumed by the NH+ 4 elimination reaction, and the decreasing concentration of the remaining NAD(P)H is monitored to calculate K+ concentrations (Table 1). This means that the K+ reaction rate may be affected by the amount of NH+ 4 in samples and reagents. (iii) The elimination system in the proposed method was simple because IMPDH was only stimulated by K+ and NH+ 4 , and was unaffected by other biological materials. By contrast, in all other enzymatic methods, one or more elimination systems besides NH+ 4 are necessary for Ca2+ or Na+ in the analyte. References
202 g/l bilirubin C, 5.00 g/l hemolytic hemoglobin, 2 mmol/l NH4Cl, 3 mmol/l LiCl, 0.05 mmol/l MnCl2, 200 mmol/l NaCl, 10 mmol/l CaCl2, or 0.1 mmol/l ZnSO4, as well as turbidity up to 1560 hormadin turbidity units. The reagents appear to be stable for at least 7 days at 4 to 8 °C and 1 month at −20 °C, as they retained their initial sensitivity under those conditions. We have developed a novel method for using IMPDH to measure K+ in serum based on increases in NADH, which is unlike all other reported enzymatic methods (Table 1) [1–3]. This proposed assay has several advantages over other enzymatic methods. (i) Because increases in NADH are being monitored, the measured range is wider than that of other methods, where the optical limits of the colorimeters reduce the range of NAD(P)H concentrations that can be measured (Tables 1 and 2). (ii) In the proposed method, the reagent conditions for K+ determination could be maintained constant, because the substrate and cofactor for the IMPDH reaction were unaffected by the NH+ 4 elimination reaction.
[1] Berry MN, Mazzachi RD, Pejakovic M, Peake MJ. Enzymatic determination of potassium in serum. Clin Chem 1989;35:817–20. [2] Kimura S, Asari S, Hayashi S, Yamaguchi Y, Fushimi R, Amino N, et al. New enzymatic method with tryptophanase for determining potassium in serum. Clin Chem 1992;38: 44–7. [3] Kimura S, Iyama S, Yamaguchi Y, Hayashi S, Fushimi R, Amino N. New enzymatic assay with urea amidolyase for determining potassium in serum. Ann Clin Biochem 1997;34:384–8. [4] Wu TW, Scrimgeour KG. Properties of inosinic acid dehydrogenase from Bacillus subtilis. II. Kinetic properties. Can J Biochem 1973;51:1391–8. [5] Ota H, Yasuda Y, Sakasegawa S, Imamura S, Tamura T. A novel enzymatic method for measuring mizoribine phosphate levels in serum. J Biosci Bioeng 2008;106:511–4. [6] Tholen DW, Linnet K, Kondratovich M, Armbruster DA, Garrett PE, Jones RL, et al. NCCLS document EP17-A: protocols for determination of limits of detection and limits of quantitation; approved guideline, vol. 24. Pennsylvania: National Committee for Clinical Laboratory Standards; 2004. [7] http://www.acb.org.uk/whatwedo/science/AMALC.aspx. [8] Kinoshita S, Toyofuku M, Iida H, Wakiyama M, Kurihara M, Nakahara M, et al. Standardization of laboratory data and establishment of reference intervals in the Fukuoka Prefecture: a Japanese perspective. Clin Chem Lab Med 2001;39:256–62.
