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Enzymatic analysis

Enzymatische Analyse

A 17 New developments of conductimetry in analytical biochemistry Patricia Duffy, Iman Saad, and Jean M. Wallach

Laboratoire de Biochimie Analytique, UA CNRS 244, Universit~ Claude Bernard-Lyon I, 43 Boulevard du t ~ Novembre 1918, F-69622 ViUeurbanne C6dex, France

Neue Entwicklungen der Leiff'ahigkeitsmessungen in der analytischen Biochemie

In the 70's, first applications of conductimetry in biochemical analysis were made with devices unsuitable for automation. In our laboratory we developed sensitive conductimetric cells to increase the potentiality of the methodology [3]. Recently, we improved our conductimetric apparatus to make them suitable for automation and data processing, and developed new enzy- matic assays.

Materials and methods

Our new titratmn cell, thermostated at 30 ~ is made of acrylic resin. It holds 5.5 ml of solution which is introduced into the cell with a peristaltic pump. Two couples of probes are inserted into the cell. One, composed of two disks of platinized platinum is for conductance measurements. The other, made of coils of platinum wire coated with glass, is linked to an electronic circuit designed for temperature regulation. Such a system keeps tem- perature within 0.01 ~ avoiding use of a circulating water-bath. The cell is connected to a conductimeter and conductance values are captured on a computer for data processing.

Results and discussion

We mainly elaborated two different assays:

Cholinesterase activity o f serum or whole blood. Methods for measurement of cholinesterase activity include potentiometry and essentially spectrophotometry [2].

We developed a rapid and simple method for both erythro- cyte and plasma cholinesterase by measurement of conductance changes during the first 2 rain of reaction.

As blood contains both acetyI- and butyrylcholinesterases, we determined the kinetic parameters of the enzymes with differ- ent substrates. The results are given in Table 1.

Considering the obtained results, for serum cholinesterase assay, 0.7 mmol/1 butyrylcholine was used as substrate. After temperature equilibrium, 5 - 1 0 gl of serum were added and conductance changes recorded.

The coefficients of within-day variation obtained were less than 5% (mean of 10 measurements). The results obtained with our conductimetric method were compared with Ellman's pro- cedure. The correlation factor was calculated statistically and found to be 0.94 (N = 40). Actually, an assay is performed within 3 - 4 min.

Table 1. Kinetic parameters of acetyl- and butyrylcholin- esterases with different substrates

Substrates AChE BuChE

Vmax KM Vmax KM (gS. h-*) (lamol/1) (ITS" h - 1) (mmol/1)

Acetyl- choline 53 10 35 0.22

Propionyl- choline 54 13 71 0.22

Propionyl- thiocholine 37 51 63 0.15

Butyryl- choline - - 88 0.14

Butyryl- thiocholine - - 86 0.14

The same methodology was used with blood. In that case, 1 mmol/1 propionylcholine was used as substrate because both acetyl- and butyrylcholinesterases had similar hydrolysis rate. For activity measurements, only 5 or 10 Ixl are needed.

The coefficient of variation was less than 5% and the corre- lation coefficient with a reference spectrophotometric assay was 0.96 (N = 50) [11.

Alcohol (ethanol) assay. By using a coupled assay with both alcohol- and aldehyde dehydrogenases, we measured the con- ductance changes mainly due to appearance of acetate ions when ethanol is oxidized.

Optimization of the assay was performed by increasing the aldehyde dehydrogenase/alcohol dehydrogenase ratio.

From the conductance changes, a kinetic method was de- veloped for alcohol assay. A linear relationship between the rate of conductance variations and alcohol concentration was obtained between 0.025 and 0.15 retool/1.

A similar kinetic assay of acetaldehyde, using only aldehyde dehydrogenase in presence of N A D + was demonstrated to be impossible because of the low KM value of the substrate for the enzyme (about 6 - 1 0 gM). So we developed an end-point method. A linear relationship between the total conductance changes and aldehyde concentration was obtained in the range 0.012-0.07 retool/l.

References

1. Augustinsson KB, Eriksson H, Faijersson Y (1978) Clin Chim Acta 89 : 239 - 252

2. Elhnan GL, Courtney KD, Andres V, Featherstone RM (1961) Biochem Pharmacol 7 : 8 8 - 9 5

3. Wallach JM (1983) Progr Clin Enzymol 2: 317 - 319

Fresenius Z Anal Chem (1988) 330:357 �9 Springer-Verlag 1988

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