Enzymic determination of glucose with SMAC: adaption of the dichlorophenol/ aminophenazone chromogen

Introduction The currently used SMAC glucose oxidase method is a modification of the procedure of Gochman and Schmitz ]. In this colorimetirc determination, the specificity of glucose oxidase is combined with a peroxidase indicator coupler, 3-methyl-2 benzothiazolinone hydrazone (MBTH) and dimethylaniline (DMA) to form a coloured indamine dye with a high molar absorption. However, as much as six different reagents have to be prepared by the user, some of them daily. Barham and Trinder [2] described in 1972 a method involving the use of 4-aminophenazone as a colour


Introduction
The currently used SMAC glucose oxidase method is a modification of the procedure of Gochman and Schmitz ]. In this colorimetirc determination, the specificity of glucose oxidase is combined with a peroxidase indicator coupler, 3-methyl-2 benzothiazolinone hydrazone (MBTH) and dimethylaniline (DMA) to form a coloured indamine dye with a high molar absorption. However, as much as six different reagents have to be prepared by the user, some of them daily. Barham and Trinder [2]  coupler with sulphonated 2,4-dichlorophenol for determining the hydrogen peroxide produced from glucose with glucose oxidase. This method had a high sensitivity and the additional possibility of using a single reagent stable for several days.
This paper presents an adaptation of the former to the SMAC system considerably reducing both the number of reagents involved and the operator-time consumed. The proposed method was compared with the hexokinase manual method and the Gochman-Schmitz method for the SMAC.

Reagents
Reagents for SMA C glucose determination (MBTH/DMA) Reagents were prepared according to the manufacturer [3].

Instrumentation
The instrument used was a SMAC (Technicon Instruments Corp, Tarrytown, NY, 10591). The flow diagram for the DCPS/AP proposed method is shown in Figure 1.   (Table 2). A number of saccharides did not interfere reflecting the specificity of the method, maltose being an exception. The interference from maltose was due to the common contamination of glucose oxidase with maltase. None of the anticoagulants tested interfered. Glutathione, uric acid, ascorbic acid and bilirubin did not interfere at concentrations higher than those found in serum. The serum drug concentrations studied here were higher than would be expected after therapeutic doses and no interference was observed. Haemoglobin did not interfere up to 50 mg/ml, but a haemolysate with the same concentration of haemoglobin produced a decrease in the observed glucose value.
Accuracy and comparison of methods Accuracy of the method was investigated by recovery studies and by comparison with the spectrophotometric hexokinase method. To investigate recovery a pool of human serum was prepared by dialysing it overnight against saline. Various amounts of glucose solution (555 mmol/1) and water were added to the dialysed serum and analysed. The results are shown in Table 3.. Recovery was excellent at low and high glucose concentration. We compared our results for this method to those obtained by the Boehringer-Mannheim hexokinase manual procedure and by the MBTH/DMA SMAC method for 85 freshly collected patients' sera. The data were correlated ( Figure 3) and showed excellent agreement over a wide range of plasma glucose values.
In conclusion, the proposed system is simpler and more economical than the MBTH/DMA method, performing similarly in linearity, sensitivity, resistance to interfering substances, accuracy and precision. Recently obtained data have shown a similar performance of the proposed method in the SMA II system modified in the glucose channel as described for the SMAC (results not shown).
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