Kinetic Measurement of Glucose with a Centrifugal Analyzer ...Clin Chem(1976)Cool!!!

Glucose oxidase reagents
Table 1 lists the conditions we used for the two procedures. Both procedures include a reagent blank in cuvette 1 (distilled water substituted for sample) and a standard in cuvette 2. The single hexokinase reagent and both components of the glucose oxidase reagent were dispensed into the reagent wells of the transfer disc. Calculations were performed with the Rotochem computer to obtain the concentration (Ca) of glucose in the serum samples (cuvettes 3-14), using the Rotochem Kinetic Rate III program as follows: CU
1 Departments of Medicine, Pathology, Biochemistry, and Hospital Laboratories, University of North Carolina, Chapel Hill, N. C. 27514, and 2 ICI Pharmaceuticals, Alderly Park, England. Received Nov. 10, 1975; accepted Mar. 25, 1976.
Within-disc precision Hexokinase
The linear range of the glucose oxidase method extended
13 13 12
888 2682 786
±8.0
±16.4 ±5.2
0.9
0.6 0.7
to 4000 mg/liter and was consistent from day to day, owing to the excellent stability of the reagents. The precision of the glucose methods was evaluated (Table 2) by performing replicate analyses on lyophilized control sera. Correlation between results by the hexokinase and glucose oxidase procedures is illustrated in Figure 1. Although the hexokinase procedure is accepted as a comparison method for
10 100
SoIn I 500
Soln 11100
35 520 60 150 Wavelength, nm Initial reading, s
the hexokinase/glucose-6-phosphate dehydrogenase (D-glucose-6-phosphate:NADP 1-oxidoreductase, EC 1.1.1.49) coupled enzyme assay (2) and the glucose oxidase (-D-glucose:oxygen oxidoreductase, EC 1.1.3.4) method of Trinder
with
1100
CLINICAL
CHEMISTRY,
Vol. 22,
No. 7, 1976
Table 2. PrecIsion of Procedures for Plasma Glucose
No.
Procedure analyses
Mean
mg/liter
SD
We selected the fixed times used for the assay on the basis of achieving a desirable balance between linearity, sensitivity, and speed of analysis. An initial reading at 5 s resulted in a decreased linear range. Extending the second reading beyond 130 s contributed little to improved sensitivity.
Table 1. Conditions for Measurement of Plasma Glucose
Hexokinase
Glucose oxidase
Sample vol, iI Flush vol, I
Reagent vols, il Temperature, #{176}C
5
160
400 30 340 10 130
where C8 = concentration of standard, LAU = change in absorbance of serum samples between the fixed times (see Table 1), LiA8 = change in absorbance of standard, and E..ARB = change in absorbance of reagent blank.
We describe automated methods for a centrifugal analyzer for plasma glucose analysis by a fixed-time kinetic approach. Results by the two methods correlated well, and their precision (day-to-day) is reflected by coefficients of variation of less than 2.5% for each method.
Results and Discussion
The linearity of the two procedures for glucose was checked aqueous glucose standards. The hexokinase method was linear to 3500 mg/liter, the upper limit of the linear range varying slightly with different vials of reagent. The linearity decreased markedly if the working reagent was kept at room temperature rather than at 0#{176}C. The upper limit of linearity could be increased to 4000 mg/liter by reducing the sample volume to 2.5 1d. Use of this small volume was satisfactory during the method-development stage, but precision was inadequate when this volume was used routinely in our service laboratory, and a 5-jl sample volume was substituted in the routine method.
Final reading, s
(3).
Materials and Methods
Apparatus.
Procedures
analyzer, the Aminco
We used
a
Co., Silver Spring, Md. 20901), an Aminco “Rotofill II”diluter being used for sample and diluent delivery. The centrifugal analyzer was programmed totransfer sample and reagents with mixing in less than 10 s. Some reagents were dispensed with Eppendorf microliter pipets (Brinkmann Instruments, Inc., Westbury,
The application of a nonlinear fixed-time kinetic approach to the hexokinase (ATP:D-hexose-6-phosphotransferase, EC 2.7.1.1) method for plasma glucose has been mentioned by Tiffany et al. (1). We have adapted and evaluated linear fixed-time kinetic methods for plasma glucose with use of both
-‘ -
1-.S
X L.AuiARB
A8
-
ARB
were
obtained
commercially
(“GOD/PAP,